.. _docs_gp_nanopore_cov: .. spelling:word-list:: artic Conda Minimap Nanopore Nanopolish basecall basecalled basecaller basecalling bp bcftools coronavirus epigenomics mRNA metagenomic nCoV nanopolish nt transcriptonomics minimap Nanopore CoVSeQ Pipeline ======================== :bdg-primary:`Version` |genpipes_version| .. include:: /common/new_gp_wizard.txt .. tab-set:: .. tab-item:: Usage .. dropdown:: Command :open: .. code-block:: user@machine:~$ genpipes nanopore_covseq [options] [--genpipes_file GENPIPES_FILE.sh] user@machine:~$ bash GENPIPES_FILE.sh .. dropdown:: Options .. include:: opt_nanopore_cov.inc .. include:: /common/gp_readset_opt.inc .. include:: /common/gp_common_opt.inc .. dropdown:: Example .. include:: /user_guide/pipelines/example_runs/nanopore_covseq.inc .. include:: /user_guide/pipelines/example_runs/nanopore-readset.inc .. include:: /user_guide/pipelines/notes/scriptfile_deprecation.inc .. card:: Test Dataset :link: docs_testdatasets :link-type: ref You can download the test dataset for this pipeline :ref:`here`. .. tab-item:: Schema :name: nanocovschema .. dropdown:: Default Figure below shows the schema of the Nanopore CoVSeQ ARTIC SARS-CoV2 sequencing protocol. You can also refer to the latest `pipeline implementation `_ .. figure:: /img/pipelines/mmd/nanopore_covseq.default.mmd.png :align: center :alt: nanopore covseq (-t default) schema Figure: Schema of Nanopore CoVSeQ (Default) Sequencing protocol .. figure:: /img/pipelines/mmd/legend.mmd.png :align: center :alt: dada2 ampseq :width: 100% :figwidth: 75% .. dropdown:: Basecalling .. figure:: /img/pipelines/mmd/nanopore_covseq.basecall.mmd.png :align: center :alt: nanopore covseq (-t basecalling) schema Figure: Schema of Nanopore CoVSeQ (Basecalling) Sequencing protocol .. figure:: /img/pipelines/mmd/legend.mmd.png :align: center :alt: dada2 ampseq :width: 100% :figwidth: 75% .. tab-item:: Steps .. dropdown:: Default +----+--------------------------------+ | 1. | |host_reads_removal| | +----+--------------------------------+ | 2. | |kraken_analysis| | +----+--------------------------------+ | 3. | |artic_nanopolish| | +----+--------------------------------+ | 4. | |wub_metrics| | +----+--------------------------------+ | 5. | |covseq_metrics| | +----+--------------------------------+ | 6. | |snpeff_annotate| | +----+--------------------------------+ | 7. | |quast_consensus_metrics| | +----+--------------------------------+ | 8. | |rename_consensus_header| | +----+--------------------------------+ | 9. | |prepare_report| | +----+--------------------------------+ .. dropdown:: Basecalling +----+--------------------------------+ | 1. | |guppy_basecall| | +----+--------------------------------+ | 2. | |guppy_demultiplex| | +----+--------------------------------+ | 3. | |pycoqc| | +----+--------------------------------+ | 4. | |host_reads_removal| | +----+--------------------------------+ | 5. | |kraken_analysis| | +----+--------------------------------+ | 6. | |artic_nanopolish| | +----+--------------------------------+ | 7. | |wub_metrics| | +----+--------------------------------+ | 8. | |covseq_metrics| | +----+--------------------------------+ | 9. | |snpeff_annotate| | +----+--------------------------------+ | 10.| |quast_consensus_metrics| | +----+--------------------------------+ | 11.| |rename_consensus_header| | +----+--------------------------------+ | 12.| |prepare_report| | +----+--------------------------------+ .. card:: .. include:: steps_nanopore_covseq.inc .. tab-item:: About .. card:: The Nanopore CoVSeQ pipeline is used to analyze long reads produced by the Oxford Nanopore Technologies (ONT) sequencers. The SOP for Nanopore data is based on the `ARTIC SARS-CoV2 protocol `_, Version 4 / 4.1 (`V4.1 `_), using nanopolish. This protocol is closely followed in GenPipes Nanopore sequencing pipeline with majority of changes related to technical adaptation of the protocol to be able to run in a High Performance Computing (HPC) environment. In such environments, Conda is not advisable. Key steps in this pipeline include `basecalling with Guppy`_, demultiplexing, read filtering and consensus sequencing. Basecalling with Guppy happens only if the ```-t basecalling``` option is selected. If basecalling protocol option is selected through the -t command line option, the Nanopore CoVSeQ pipeline will do `basecalling with Guppy`_ (GPU) and demultiplexing. After basecalling, the pipeline performs de-hosting, for all the samples, followed by running the ARTIC-Nanopolish wrapper which performs alignment to the SARS-CoV2 reference (using `minimap2 `_), variant calling (using `Nanopolish software `_). The Nanopolish software performs signal-level analysis of Oxford Nanopore sequencing data. After Nanopolish processing, the pipeline performs consensus generation through artic_mask and bcftools consensus steps. Lastly, custom scripts and ncov_tools are run to report on quality metrics for Nanopore CoVSeQ GenPipes Sequencing Pipeline. Details of structure and contents of the `Nanopore readset file are available here `_. See See :ref:`nanocovschema` tab for the pipeline workflow. For more details, refer to the `README file `_ file. **References** * `nCoV-2019 novel coronavirus bioinformatics protocol`_ * `Phylogenetic Analysis of nCoV-2019 genome`_ using publicly shared genome sequences with datasets from NCBI or GISAID. * `Tiling Amplicon sequencing and downstream bioinformatics analysis`_ .. include:: /user_guide/pipelines/notes/artic_version.inc .. The following are replacement texts used in this file .. |guppy_basecall| replace:: `Guppy Basecall`_ .. |guppy_demultiplex| replace:: `Guppy Demultiplex`_ .. |pycoqc| replace:: `pycoQC`_ .. |host_reads_removal| replace:: `Host Reads Removal`_ .. |kraken_analysis| replace:: `Kraken Analysis`_ .. |artic_nanopolish| replace:: `ARTIC Nanopolish`_ .. |wub_metrics| replace:: `Wub Metrics`_ .. |covseq_metrics| replace:: `CoVSeQ Metrics`_ .. |snpeff_annotate| replace:: `SnpEff Annotate`_ .. |quast_consensus_metrics| replace:: `Quast Consensus Metrics`_ .. |rename_consensus_header| replace:: `Rename Consensus Header`_ .. |prepare_report| replace:: `Prepare Report`_ .. The following are links and references used in this file .. _Nanopore ARTIC-Nanopolish protocol: https://artic.network/ncov-2019 .. _Phylogenetic Analysis of nCoV-2019 genome: https://virological.org/t/phylodynamic-analysis-176-genomes-6-mar-2020/356 .. _nCoV-2019 novel coronavirus bioinformatics protocol: https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html .. _Tiling Amplicon sequencing and downstream bioinformatics analysis: https://artic.network/quick-guide-to-tiling-amplicon-sequencing-bioinformatics.html .. _basecalling with Guppy: https://timkahlke.github.io/LongRead_tutorials/BS_G.html .. _Wub Package: https://github.com/nanoporetech/wub .. _Guppy: https://timkahlke.github.io/LongRead_tutorials/BS_G.html